Eucaryotic chromosomes end in specialized structures, called telomeres [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939] that are thought to fulfill at least three functions. First, telomeres protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock, Genetics, 26:234-282 (1941)]. Second, cytogenetic observations indicate that telomeres are located at the nuclear periphery, suggesting a role for chromosome ends in the architecture of the nucleus [Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J., 10:214-330 (1885)]. Third, telomeres must provide a solution to the end-replication problem [Watson, Nature, 239:197-201 (1972)]: because all known polymerases require a primer and synthesize DNA from 5' to 3', the 3' ends of linear DNA pose a problem to the replication machinery.
The single common structural feature of most eucaryotic telomeres is the presence of a tandem array of G-rich repeats which, according to genetic studies in Saccharomyces cerevisiae, are necessary and sufficient for telomere function [Lunablad et al., Cell, 83:633-643 (1989); Szostak et al., Cell, 36:459-568 (1982)]. Although all telomeres of one genome are composed of the same repeats, the terminal sequences in different species vary. For instance, Oxytricha chromosomes terminate in TTTTGGGG repeats [Klobutcher et al., Proc. Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG).sub.n [Blackburn et al., J. Mol. Biol., 120:33-53 (1978)], plant chromosomes carry the sequence (TTTAGGG).sub.n (Richards et al., Cell, 53:127-136 (1988)], and trypanosomas and mammals have TTAGGG repeats at their chromosome ends [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature, 338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis et al., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg et al., Cell 36:459-468 (1984)]. The organization of the telomeric repeats is such that the G-rich strand extends to the 3' end of the chromosome. At this position, telomerase, an RNA-dependent DNA polymerase, first demonstrated in Tetrahymena thermophila and other ciliates, can elongate telomeres, probably by using an internal RNA component as template for the addition of the appropriate G-rich sequence [Greider and Blackburn, Cell, 43:405-413 (1985)]. This activity is thought to complement the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al., Nature, 337:429-433 (1989)]. Recently, it has been reported that the addition of telomerase into a cultured human cell leads to an increase of the proliferative life-span of that cell [Bodner et al., Science, 279:349-352 (1998)].
Proximal to the essential telomeric repeats, some chromosome ends harbor additional common elements called sub-telomeric repeats or telomere-associated sequences [Chan et al., Cell, 33:563-573 (1983); Corcoran et al., Cell, 53:807-813 (1988); de Lange et al., Nucl. Acids. Res., 11:8149-8165 (1983); Van der Ploeg et al. (1984); Dunn et al., Cell, 39:191-201 (1984)]. Unlike telomeric repeats, these sequences are not conserved and their function remains unclear [Murray et al., Mol. Cell. Biol., 6:3166-3172 (1986)].
Chromosome ends of unicellular organisms often show structural instability. Frequent rearrangements of subtelomeric sequences occur in trypanosomas [Borst, Annu. Rev. Biochem., 55:701-732 (1986), de Lange et al. (1983)], S. cerevisiae [Carlson et al., Mol. Cell. Biol., 3:351-359 (1983); Horowitz et al., Mol. Cell. Biol., 4:2509-2517 (1984)], and plasmodia [Corcoran et al., (1988)], and changes in the telomeric repeat region can be observed in protozoa [Bernards et al., Nature, 303:592-597 (1983); Pays, Nucl. Acids. Res., 11:8137-8147 (1983); Van der Ploeg (1984)], ciliates [Larson et al., Cell, 50:477-483 (1987)], and fungi [Carson et al., Cell, 42:249-257 (1985); Lundblad et al., (1989); Lustig et al., Proc. Natl. Acad. Sci. USA, 83:1398-1402 (1986)]. As much as 3.5 kilobase pairs (kb) was seen to be added to telomeres of Trypanosoma brucei in a process that appears gradual and continuous, and was calculated to result from the addition of 6 to 10 base pairs (bp) per end per cell division [Bernards et al., (1983); Pays et al., (1983); Van der Ploeg, (1984)]. A similar gradual telomere elongation, compatible with the addition of telomeric repeats by telomerase, occurs in continuously growing T. thermophila [Larson, (1987)] and a cell cycle mutant (cdc17) of S. cerevisiae [Carson et al., (1985)]. In wild-type S. cerevisiae [Shampay et al., Proc. Natl. Acad. Sci. USA, 85:534-538 (1988)], however, and in T. thermophila grown in batch cultures [Larson et al., (1998)], the tandem array of telomeric repeats is maintained at constant length. At least four genes (CDC17, EST1, TEL1, and TEL2 [Carson et al., (1985); Lundblad et al., (1989); Lustig et al., (1986)] govern the length and stability of yeast telomeres; their mode of action is not understood.
Much less is known about the structure and behavior of chromosome ends of multicellular organisms. Mammalian telomeres have become amenable to molecular dissection with the demonstration that telomeric repeats of plants and T. thermophila species cross-hybridize to vertebrate chromosome ends [Allshire et al., Nature, 332:656-659 (1988); Richards et al., (1988)]. It has also been shown that human DNA contains tandem arrays of TTAGGG repeats, probably at the chromosome ends, providing further evidence for the evolutionary conservation of telomeres and a tool for the isolation of telomeric DNA [Moyzis et al., (1988)]. Two strategies to obtain human chromosome ends have proven successful: an indirect isolation protocol that relies on human telomeres to be functional in S. cerevisiae [Brown et al., (1989); Cross et al., (1989)] and direct cloning in E. coli.
de Lange et al. [Mol. Cell. Biol., 10:518-527 (1990)] characterized the structure and variability of human autosomal chromosome ends. The chromosome ends they analyzed shared a sub-telomeric repeat of at least 4 kb that is not conserved in rodent genomes. These chromosome ends were characterized by a long stretch of DNA, of up to 14 kb, that lacks restriction enzyme cutting sites and may be entirely composed of TTAGGG repeats. From this region sequences are lost during development, leading to shortened, heterogeneously sized telomeres in somatic tissues, primary tumors, and most cell lines.
de Lange [EMBO J., 11:717-724 (1992)] reported that human telomeres are tightly associated with the nuclear matrix. Telomeres were demonstrated to be anchored via their TTAGGG repeats. Moreover, TTAGGG repeats at internal sites within the chromosome do not behave as matrix-attached loci, suggesting that the telomeric position of the repeats is required for their interaction with the nuclear matrix. This evidence is consistent with the role of telomeres in a nucleoprotein complex.
TRF activity was first identified in 1992 by Zhong et al. [Mol. Cell. Biol, 12:4834-4943 (1992)] as a DNA-binding factor specific for TTAGGG repeat arrays. TRF was found to be present in nuclear extracts of human, mouse and monkey cells. The optimal site for TRF binding was found to contain at least six contiguous TTAGGG repeats. However, the protein isolated by Zhong et al. was not sufficiently purified from other DNA-binding proteins such that its amino acid sequence could be determined.
Saltman et al. [Chromosoma, 102:121-128 (1993)] characterized the molecular structure of telomeres of two human tumor cell lines with frequent end-to-end associations of metaphase chromosomes. Such end-to-end associations have been observed in a variety of human tumors, aging cells and several chromosome instability syndromes. The telomeres of such end-associated chromosomes were shown by Saltman et al. to be severely reduced compared to other human cells with functional telomeres. However, other cell lines with severely shortened telomeres were not detectably compromised in their function. Thus, the investigators suggested that telomeric length was not the only determinant of the fusigenic behavior of human telomeres in tumor cells.
A Xenopus laevis protein factor that specifically recognizes vertebrate telomeric repeats at DNA ends, termed Xenopus telomere end factor (XTEF) was identified by Cardenas et al. in 1993 [Genes and Devel., 7:883-894 (1993)]. The DNA-binding properties of XTEF resembled the characteristics of a class of terminus-specific telomere proteins identified in hypotrichous ciliates.
There has been speculation on the role of an enzyme termed telomerase in human cancer, in particular in ovarian carcinoma [de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Telomerases use the 3' end of DNA as a primer and employ an RNA template for the synthesis of G-rich telomeric repeats. Telomerase activation appears to be an obligatory step in the immortalization of human cells.
Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the investigators reported that the sequence dependence of telomere formation matched the in vitro binding requirements for TRF1.
Although the activity of TRF1 had been identified and isolated to some extent, the purification of TRF was fraught with difficulty, both in isolating the protein away from other DNA binding proteins, and in obtaining active protein from the isolate.
Therefore, there is a need to isolate and characterize vertebrate TRF1. In addition, there is a need to identify other vertebrate telomere repeat binding factors which must also serve as structural and/or functional proteins in the maintenance of normal telomere physiological processes. Further, there is a need to isolate and characterize such TRFs (including TRF2) and to distinguish their characteristics from TRF1, as well as ascertain the role such TRFs play in telomere maintenance and elongation.